Binding sites ethidium

Scheme 2 Mechanism for the binding of ethidium cation (G) to two binding sites of DNA (A and B). For the intramolecular conversions, kAB and kBA are bimolecular rate constants where (G-DNA)a or (G-DNA)b react with a second DNA molecule leading to transfer of the guest.

K e = 22 x 10s M Linear pBR322 remains uniform and independent of added chloroquine up to chl/bp = 25. The decline in the amplitude ratio at large chl/bp is due to competition between ethidium and chloroquine for binding sites on the DNA and is used to determine the chloroquine binding constant. 

Since tRNA is more varied structurally than DNA, ethidium could reside in pockets as well as intercalate into double-strand regions. The fluorescence decay provides information about the type, or types, of binding sites occupied by ethidium. It is currently believed that the excited state of ethidium is quenched by proton transfer to the solvent0 86-1 and that its lifetime is reduced with increasing solvent exposure. If ethidium occupies two or more kinds of sites with different degrees of exposure to solvent, then its fluorescence decay is expected to be multiexponential. 

Satisfactory fits of the fluorescence decays for ethidium bound to yeast tRNAPhe and E. coli tRNA al require (at least) two exponentials in the sum response S(t) [cf. Eq. (4.56)] under all conditions studied.0 870 88) The normalized amplitudes and lifetimes for tRNA 1 (extrapolated to zero concentration) are S° = 0.917, r, = 25.6ns and S02 = 0.082, t2 = 5.6 ns.(187) The results for tRNAPhe are similar.(188) This requirement for two (or more) exponentials is unequivocal evidence for at least two ethidium binding sites. The dominant component has a lifetime similar to, but slightly longer than, that of ethidium intercalated in DNA and is taken to represent ethidium... 

Students will follow the absorption and fluorescence spectral modifications of ethidium bromide in the presence of different DNA concentrations. Then, they will calculate the number of binding sites and the mean association constant. Before coming to the lab, students should determine the absorption and emission spectra of ethidium bromide bound to DNA. 

From the absorption spectrum of bound DNA you find in the literature, can you tell if there are any wavelengths where bound ethidium bromide does not absorb Also, from the absorption spectra you recorded, are there any wavelengths where bound ethidium bromide does not absorb or its absorption is weak Plot at this wavelength the OD of ethidium bromide as a function of added DNA concentration. Use the data to determine the number of binding sites and the association constant of the complex. Compare the results obtained in 3 and 4. 

The fluorescence intensity of ethidium bromide increases in the presence of DNA (Figure 12.2). Once saturation is reached, the intensity increase stops. This intensity increase means that complex formation is occurring. The intensity increase is proportional to the number of binding sites and to the affinity constant. 

Figure 12.7 Titration of 50 /xM ethidium bromide with herring DNA. The number of binding sites is equal...

Ethidium Switches the Nuclease-Detectable Binding Sites of cis-DDP on DNA... 

In examining further this effect of ethidium, we found that the exonuclease III method described above detected different cis-DDP binding sites on a DNA molecule of known sequence when platination was carried out in the presence of ethidium (56). 

Figure 7. Autoradiograph of an 8% polyacrylamide/7 M urea electrophoresis gel showing the effect of ethidium bromide on exonuclease Ill-detected cis-DDP binding sites on a 165-base-pair DNA molecule.

The DNA-binding affinities of metallo-MPE were obtained with inert metal ions. The affinity constants of Ni -MPE and Mg -MPE are 2.4 X 10 and 1.5 X 10 M, respectively, similar to the affinity constant of ethidium bromide (125). As expected for an intercalator, Mg -MPE unwinds DNA (11 3° per bound molecule), and the binding site size is two base pairs. 

Heat the mixture to 85 C for 2 min, transfer to a 70 C water bath and let the bath cool to room temperature over several hours. The extent of hybridization can be checked on a native agarose or acrylamide gel (by ethidium bromide staining). The annealed DNA is stored at 4 C. Before proceeding to the coupling reaction, the effectiveness of the annealed oligonucleotides to bind to the particular protein should be checked by competition with a labeled binding site in a DNA-binding assay. 

Ultrasonic methods have been used to study the syn anti equilibrium of 2-deoxyadenosine in presence of ethidium bromide and indole-3-acetic acid, which, respectively, serve as models for an intercalating drug and a tryptophan unit at a protein binding site. ... 

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